Many disease processes involve alterations in the chemical makeup of tissue. Synchrotron-based infrared (IR) and x-ray fluorescence microscopes (XRFM) are becoming increasingly popular for imaging both organic and trace metal compositions of biological cells and tissues, respectively, without the need for extrinsic labels or stains. Coupled with visible light microscopy, these techniques can be used to correlate conventional histological structure to organic and trace-element composition. Fourier transform infrared microspectroscopy (FTIRM) provides chemical information on the organic components of a material at a diffraction-limited spatial resolution of 2-10 μm in the mid-infrared region. FTIRM provides chemical information on the organic components of a tissue such as proteins, lipids, nucleic acids, and carbohydrates. FTIRM is very sensitive to protein secondary structure in tissue as well, where the frequency of the Amide I band, assigned to the amide (>C═O) backbone of the protein, has a different absorption maximum for α-helical (˜1655 cm−1), β-sheet (˜1630 cm−1), and extended coil (˜1645 cm−1) proteins. For example, the structure of misfolded protein aggregates has been identified in the brain tissue of Alzheimer's disease patients and infectious prion proteins have been characterized in scrapie, a sheep's form of mad cow disease. In addition to protein structure, variations in bone composition have been observed in osteoporosis, osteopetrosis, and osteoarthritis. In heart disease, altered lipid and collagen content and structure in the myocardium have been seen, which were partially normalized by losartan treatment.
Synchrotron x-ray fluorescence (SXRF) microprobe is used to probe trace elements with sensitivities, for example, in the sub-mg kg−1 range and a spatial resolution similar to FTIRM (2-10 μm). Because of the low power deposition that x-rays provide and the ability to conduct the analyses in air, these analyses can be done non-destructively on a much wider array of sample types, especially, relatively fragile biological samples. For example, the alterations in trace metals such as Fe, Cu, and Zn have been observed in neurological diseases such as Parkinson's disease, amylotrophic lateral sclerosis, Alzheimer's disease, and prion diseases and cancer. Environmental toxins have also been imaged in human tissue, such as elevated levels of methyl mercury and lead in hair.
In many disease states and environmental contamination, both the organic and metal ion compositions are altered. Therefore, in order for all imaging techniques applied to a single sample to be most beneficial as analytical tools, it is desirable to combine their results, which requires precise overlap of the visible, IR and x-ray images. Yet most of the abovementioned studies utilize only a fraction of the available tools, and therefore, do not examine every aspect of the disease. As a result, there can be pertinent information that is missed regarding the relationship between the alterations of the organic and metal contents, which plays a vital role in understanding the origins or other aspects of the diseases.
One means of remedying this situation is to register one image space to another image space. The goal of registering two (or more) separate and arbitrarily oriented images is to align the coordinate systems of the two images such that any given point in the scanned biological sample is assigned identical addresses in both images. The calculation necessary to register the two coordinate systems requires knowledge of the coordinate vectors of at least three points (for a 3-D image space) in the two systems. These points are referred to as “fiducial points” or “fiducials,” and the fiducials used are the geometric centers of markers, which are referred to as “fiducial markers”. The fiducials are used to correlate image space to another image space, or to correlate image space to a physical space. The fiducial markers provide a constant frame of reference visible in a given imaging mode to make registration possible.
One problem recognized as extant is the provision of fiducials capable of use with more than one imaging modality. For example, in the case of computed tomographic imaging (CT) and magnetic resonance imaging (MRI), the bony structure information from a CT scan could be integrated with soft tissue anatomical information from an MRI scan. MRI and x-ray CT images are digital images that are formed point by point. Collectively the points are called picture elements, or pixels, and are associated with an intensity of light emitted from a cathode ray tube, or are used to form an image on film. The manner in which the intensity of any given pixel is altered or modulated varies with the imaging modality employed. In x-ray CT, the modulation is, in general, a function of the number of electrons per unit volume being scanned. While in MRI, the parameters largely influencing this modulation are the proton spin density and longitudinal and transverse relaxation times T1 and T2, which are also known as the spin-lattice and spin-spin relaxation times, respectively.
In another example, XRFM is performed by focusing a small x-ray beam (about 10 microns square) on a sample and raster-scanning the sample through the beam to collect an x-ray fluorescence spectrum at each pixel. By integrating the fluorescence intensity of a particular trace metal at each pixel, a metal-distribution image can be generated. Currently, with XRFM, a light microscope objective is used to view the sample. However, the light microscope objective is unable to visualize the actual location of the x-ray beam.
In constructing a fiducial marker, it must be taken into consideration that an object that can be imaged under one imaging modality will not necessarily be imageable under another modality. Another reason for precise overlap of images is in the case of differing reflective optics such that their corresponding images are not correlated. Specifically, with XRFM, in which images are not correlated, visible light illumination and sample magnification is performed through a glass microscope objective. The visible light image and x-ray beam must be precisely overlapped in order for the XRFM image to align with the light microscope image. This can be a challenge as any motion in the x-ray beam or the visible light optics can prevent precise overlapping. Further, the lack of alignment in the images is not detected during data analysis and collection because the x-ray beam is not visible.
The ability to image with at least one imaging technique and correlate that image with a light microscope image would be particularly useful because images derived from different imaging modalities could then be registered and analytical tools such as, for example, XRFM can be used more efficiently when coupled with for example FTIRM. Therefore, there remains a need for fiducial markers that can be used to correlate at least one analytical image produced by at least one imaging modality with a viewing image of a microscope objective or with a second analytical image. To achieve this end, the present invention relates to a fiducial marking grid which can correlate at least one analytical image with a light microscope image or with a second analytical image.